John M. S. Bartlett.pdf - Bio-Nica.info

112 Wang and Young
2. First-strand cDNA synthesis: same as above (see Subheading 3.2.).
3. Second-strand cDNA synthesis: same as above (see Subheading 3.2.). T4 DNA Polymerase
was not used because blunt ending of cDNA was unnecessary in this case.
4. Purification of double-stranded cDNA: same as above (see Subheading 3.2.).
5. Primer design for PG10.2 3′-RACE: A sense gene-specific primer (SGSP of PG10.2,
5′-GAGTGAGAAGCAAAGTGCAAATGCC-3′) was designed based on the known 145 bp
sequence information of PG10.2. An anchor primer (5′-CCAAGCTATTTAGGTGACAC-
TATAGGCCATCGAGGCC-3′) was designed to prime at the end of the newly synthesized
second-strand cDNA (Fig. 2). This end sequence on the newly synthesized second-strand
cDNA is complementary to the 5′ tailing part of the oligo-dT primer used for first-strand
cDNA synthesis.
6. PCR was performed as described (see Subheading 3.2.) with SGSP of PG10.2 and the
above-mentioned anchor primer. The PCR product was electrophoresed on a 1% agarose
gel. A 4-kb fragment 3′ to the 145 bp known sequence was amplified very efficiently by
this 3′-RACE PCR (Fig. 4, lane 4).
7. The band was purified (as in Subheading 3.2.) and ligated into pGEM-T vector (as in
Subheading 3.2.).
3.4. 5′-RACE Using Gene-Specific Primer for cDNA Synthesis
(see Note 9)
1. To clone the full-length cDNA for the large transcript (8.0 kb mRNA) of PG10.2, a
gene-specific RACE cDNA was generated and used as described below.
2. First-strand cDNA synthesis was performed essentially as described (see Subheading 3.2.)
except that an antisense gene-specific primer (AGSP1 of PG10.2, 5′-TTCAAGGGCCAGT-
CAGGCCGTAGGTCACAGACACTTTGAC-3′) based on 145-bp sequence information
for PG10.2 was used for first-strand cDNA synthesis. The cDNA generated by this cDNA
synthesis primer (GSP) is only suitable for 5′-RACE of this specific gene.
3. Second-strand cDNA synthesis: same as above (see Subheading 3.2).
4. Purification of double-stranded cDNA: same as above (see Subheading 3.2).
5. Adaptor ligation: same as above (see Subheading 3.2.).
6. Primer design for PG10.2 5′-RACE: An antisense gene-specific primer2 (AGSP2,
5′- GGCAGTTCATCCACACTCAGGTACCCAG-3′) based on 145 bp sequence information
was designed for 5′-RACE of PG10.2. Notably, this primer was designed to be
upstream of the AGSP1 used for cDNA synthesis (see Subheading 3.4.). In this design,
AGSP2 is a nested primer relative to AGSP1 (Fig. 2).
7. PCR was performed as described (see Subheading 3.2). Agarose gel electrophoresis
showed that the AGSP2 of PG10.2 coupled with AP1-linker primer (Clontech, complementary
to the sequence of the ligated-adaptor) amplified a unique band of about 3.5 kb
in size upstream of the 145-bp known sequence of the PG10.2 fragment (Fig. 2, lane1).
When only the AGSP2 of PG10.2 or AP-1 was used in PCRs, this faint but sharp band
disappeared (Fig. 4, lanes 2 and 3).
8. The band was purified (see Subheading 3.2.) and ligated into pGEM-T vector (as in
Subheading 3.2.).
9. The 4368-bp sequence, including the entire 5′-RACE product and the 5′-portion of the
3′-RACE product, was determined (Fig. 4 in ref. 7).
4. Notes
1. Template purity (free of DNA) and integrity (minimum degradation) are critical for
effective isolation of full-length cDNA of interest. One should place the dissected
tissue immediately on dry ice before isolating total and poly (A)+ RNAs. All standard